Candida
spp.: manual identiication (reference method) and automated
identiication (Vitek system platform)
Mondelli AL (1), Niéro-Melo L (1), Bagagli E (2), Camargo CH (2), Bruder-Nascimento A (2), Sugizaki MF (2), Carneiro MV (1), Villas Boas PJF (1)
(1) Department of Internal Medicine, Botucatu Medical School, São Paulo State University (UNESP – Univ Estadual Paulista), Botucatu, São Paulo State, Brazil; (2) Department of Microbiology and Immunology, Botucatu Biosciences Institute, São Paulo State University (UNESP – Univ Estadual Paulista), Botucatu, São Paulo State, Brazil.
Abstract: Yeasts are becoming a common cause of nosocomial fungal infections that afect immunocompromised patients. Such infections can evolve into sepsis, whose mortality rate is high. This study aimed to evaluate the viability of Candida species identiication by the automated system Vitek-Biomerieux (Durham, USA). Ninety-eight medical charts referencing the Candida spp. samples available for the study were retrospectively analyzed. The system Vitek-Biomerieux with Candida identiication card is recommended for laboratory routine use and presents 80.6% agreement with the reference method. By separate analysis of species, 13.5% of C. parapsilosis samples difered from the reference method, while the Vitek system wrongly identiied them as C. tropicalis, C. lusitaneae or as Candida albicans. C. glabrata presented a discrepancy of only one sample (25%), and was identiied by Vitek as C. parapsilosis. C. guilliermondii also difered in only one sample(33.3%), being identiied as Candida spp. All C. albicans, C. tropicalis and C. lusitaneae samples were identiied correctly.
Key words: candidemia, Candida spp., identiication, automated identiication.
S
H
O
R
T
C
O
M
M
U
N
IC
A
T
IO
N
INTRODUCTION
A global increase in yeast incidence has been reported by several authors over the past 20 years. Among invasive infections caused by Candida spp., the blood infections known as candidemias are the most clinically relevant (1). Candida spp. is currently among the ive main causes of blood infections in hospitals, and the increase in candidemia cases has been noted mainly among patients using antibiotics, under immunosuppressive therapy or parenteral nutrition and patients subjected to multiple invasive procedures (2, 3). Some studies estimate mortality atributed to Candida spp. to be 38%, although it can vary from 50 to 60% (4-7). With regard to epidemiological aspects, identifying the yeast species is essential for monitoring hospital infection rates and for early
identiication of Candida infection outbreaks (8). herefore quickly and accurately identifying the yeast species and treating the patient as soon as possible are all fundamental measures to increase survival.
he aim of this study was to comparatively evaluate diferent methods for identifying Candida spp.
used in this study were stored in nutrient broth with cryopreservatives at –70°C, in the Cultures Collection at the Department of Microbiology and Immunology (at the Botucatu Biosciences Institute, UNESP).
Sample size was calculated using the formula of Fisher and Belle, with a 95% conidence interval and 5% precision for expected prevalence of fungemia patients. We used as reference the study of Beck-Sagué and Jarvis (9), which had 10.4% fungemia patients.
he growth medium used for yeast detection was Bactec hemoculture vials (Aerobic, Peds or Myco/F, BD – Becton Dickinson, USA); for isolation and identiication were used Sabouraud dextrose agar 2%, Columbia blood agar and Chromagar Candida (Difco, USA).
Manual Identiication Method
Yeast species were identiied according to morphological and biochemical characteristics. Ater isolation from hemoculture vials, tests were performed to verify germinative tube and chlamydoconidium production and ilamentation, followed by tests of carbon and hydrogen assimilation and carbohydrates fermentation, as described below, following the methods prescribed by Kurtzman and Fell (10).
Carbon and hydrogen assimilation
A carbon auxanogram was used to evaluate the capacity to assimilate a diferent carbon source. Yeast suspension was incorporated into a synthetic base free of carbon and solidiied. Ater that, 16 diferent carbohydrates were added to the plate, which was then incubated at 35°C for 24 and 48 hours. Any visible growth, indicated by turbidity on the spot of carbohydrate incorporation was deined as constituting positive assimilation.
Fermentation of Carbohydrates (Zymogram)
he capacity of yeasts to use certain carbohydrates as an energy source to produce ethanol and carbon dioxide (CO2) was analyzed under low oxygen tension. CO2 production could be observed in a liquid growth medium with one carbohydrate and an inoculum with 6 carbohydrates in Durham tubes. Results were analyzed at 24, 48 and 72 hours and at 5, 10 and 14 days. his was considered the reference methodology.
Automated Identiication Method
We used Vitek I (Durham, USA) as the automated identiication method, which uses a plate with 40 diferent tests of carbohydrates and amino acids, incubated at 35°C for 24 to 48 hours. Data obtained were sorted into charts as well as frequency and association tables. Numeric variables were presented as mean and standard deviation, when variables presented symmetrical (normal) distribution, or median and percentiles, in case of asymmetrical distribution. Categorical variables were expressed by number and rate of events and evaluated by chi-square test or Fisher’s exact test (if n < 10). Normal distribution variables were evaluated by Student’s t test and non-normal variables by Wicoxon-Mann-Whitney test. ANOVA (analysis of variance) was used to compare three groups. Results were considered signiicant if p < 0.05. A concordance test was used to analyze laboratory assays.
his study was approved by Botucatu Medical School’s Research Ethics Committee on October 2nd, 2006 (protocol number 2266/2006).
Using the reference method we identiied in 98 samples: 37 Candida parapsilosis; 33 Candida albicans; 12 Candida spp.; 7 Candida tropicalis; 4 Candida glabrata; 3 Candida guilliermondii and 2 Candida lusitaneae.
Table 1 shows the concordance rate between the two identiication methods evaluated (manual identiication, considered the reference method, and automated identiication, using the Vitek system) for Candida species identiication. here was a global concordance of 79 samples (80.6% of cases). Separate analysis of species found that ive samples of C. parapsilosis difered from the reference method (13.5%); Vitek wrongly identiied one of these ive samples as C. tropicalis, one as C. lusitaneae and three others as C. albicans. C. glabrata difered only in one sample (25%), which was identiied by Vitek as C. parapsilosis. One C. guilliermondii sample was wrongly identiied by Vitek as Candida spp. (33.3%). All samples identiied as C. albicans, C. tropicalis or C. lusitaneae by the manual method were identiied correctly by Vitek. Candida spp. samples difered in 12 samples (91.6%), identiied as C. albicans (six of them), C. parapsilosis (four of them) and C. tropicalis (one of them), and one was wrongly identiied as Cryptococcus neoformans.
of patients who present symptoms of blood infection, hemoculture is a low-sensitivity marker, with an elevated false-negative rate that hampers the diagnosis of this disease (11-14). his makes it diicult to decide when treatment should begin. On average, it takes from 2.1 ± 1.3 days to 5.1 ± 3 days to initiate treatment ater obtaining a positive hemoculture, and many cases are only diagnosed during autopsy.
Candida spp. grows on regular growth media but positivity is very low even when the concentration is high. In premature patients the volume of blood used for hemocultures can be the cause of low sensitivity. here is a 65% chance of detecting bacteria when 1 mL of blood is used, but low concentrations and low replication rates can reduce growth rates in culture media, thereby increasing the time necessary to obtain positivity. Negative hemocultures do not exclude diagnosis, so serial samples must be analyzed (11-13).
Candida spp. identiication and antifungal sensitivity tests are fundamental for therapeutical success. he choice of the Vitek I system platform as the automated method used for this study was based on the following reasons: a concordance rate above 80% when compared to the reference method; identiication and antifungal sensitivity tests are performed in just a few hours, being
useful for laboratories with great demand of said tests; it is able to identify Candida dubliniensis; this system presents low costs, when compared to other methods such as molecular biology techniques, and it also ofers greater safety for operators and reduces repetitive manual operations (15, 16).
C. glabrata is not very sensitive to luconazole, and Candida krusei is intrinsically resistant to luconazole; therefore, they must be treated with amphotericin B or an echinocandin. C. lusitaniae is resistant to amphotericin B, being treated with luconazole. In this context, the choice of the correct antifungal drug is delayed until identiication and sensitivity tests are performed. Yeast species identiication is also important for monitoring rates of hospital infection, and for early identiication of Candida infection outbreaks (8).
he use of diferent identiication methods and sensitivity tests allows us to verify the quality of the results more accurately. When comparing the methods of identifying Candida spp., it becomes clear that the most efective tests are also the more time-consuming and laborious ones, which makes it very important that the laboratory routine have the ability to compare and correlate these methods, in order to choose Table 1. Comparison between manual and automated (Vitek I) identiication
Manual identiication
Vitek
Total
C. pa
ra
psilosis
C. albic
a
ns
C. glabr
a
ta
C. tropic
alis
C. lusit
a
neae
C. guilliermondii C
a
ndida
spp
.
Cr
ypt
o
co
ccus
ne
oforma
ns
C. parapsilosis 32 3 0 1 1 0 0 0 37
C. albicans 0 33 0 0 0 0 0 0 33
C. glabrata 1 0 3 0 0 0 0 0 4
C. tropicalis 0 0 0 7 0 0 0 0 7
C. lusitaneae 0 0 0 0 2 0 0 0 2
C. guilliermondii 0 0 0 0 0 2 1 0 3
Candida spp. 4 6 0 1 0 0 0 1 12
Total 37 42 3 9 3 2 1 1 98
the best one. his study analyzed two methods of Candida identiication: one manual and more complex (morphological and biochemical tests), considered a reference method for many years before the introduction of molecular biology; and an automated method, using Vitek-Biomerieux (Durham, USA), which is faster and simpler.
When comparing these two distinct methods of Candida identiication we can observe a global concordance of 80.6%. he concordance rate for C. albicans (n = 33), C. lusitaneae (n = 2) and C. tropicalis (n = 7) was 100%. he concordance rates for other species were the following: C. parapsilosis – 86.49%; C. glabrata – 75% and C. guillermondii – 66.67%. Even though the concordance rate for Candida spp. was 0%, that can be explained by the fact that there is no such option on the Vitek system, what makes the system choose the most viable option, even if it is not necessarily the most accurate. hese data allow us to conclude that the Vitek-Biomerieux semi-automated method can be used in the laboratory routine, although identiication of certain species may require conirmation by another method.
Candida species peculiarities justify the need to identify yeast species when they are related to systemic diseases. his is essential for the choice of the best therapeutical approach for the patient. C. krusei isolates are totally resistant to luconazole, while C. glabrata samples are oten either resistant or not very sensitive to azoles, requiring a higher dose for treatment success. Higher doses of amphotericin B should also be used to treat invasive infections by C. krusei and C. glabrata, whereas C. lusitaniae isolates can be resistant to this drug (17, 18).
A fast and accurate technique for yeast identiication is very important for microbiological laboratories. According to the results found in the present study, the automated system Vitek-Biomerieux (Durham, USA) with an identiication card is recommended for use in routine laboratories.
ACKNOWLEDGMENTS
he authors are grateful for the support of FAMESP; the Clinical Hospital and Clinical Analysis Laboratory of Botucatu Medical School, and the Department of Microbiology and Immunology of the Botucatu Biosciences Institute, UNESP, Botucatu, SP.
COPYRIGHT
© CEVAP 2012
SUBMISSION STATUS
Received: March 28, 2012.
Accepted: June 15, 2012.
Abstract published online: June 29, 2012.
Full paper published online: August 31, 2012.
CONFLICTS OF INTEREST
he authors declare no conlicts of interest.
FINANCIAL SOURCE
he Medical and Hospital Development Foundation (FAMESP) provided the inancial grants.
ETHICS COMMITTEE APPROVAL
he present study was approved by the Research Ethics Committee of the Botucatu Medical School, UNESP, under protocol number 2266/2006 – CEP.
CORRESPONDENCE TO
Alessandro Lia Mondelli, Departamento de Medicina Interna, Faculdade de Medicina de Botucatu, UNESP, Distrito de Rubião Júnior, s/n, 18618-970, Botucatu, SP, Brazil. Phone: +55 14 3811 6213. Email: [email protected] or [email protected].
REFERENCES
1. Colombo A, Guimarães T. Epidemiology of
hematogenous infections due to Candida spp. Rev Soc
Bras Med Trop. 2003;36(5):599-607.
2. Nucci M, Colombo A. Risk factors for breakthrough candidemia. Eur J Clin Microbiol Infect Dis. 2002;21(3):209-11.
3. França J, Ribeiro C, Queiroz-Telles F. Candidemia em um hospital terciário brasileiro: incidência, frequência das diferentes espécies, fatores de risco e suscetibilidade aos antifúngicos. Rev Soc Bras Med Trop. 2008;41(1):23-8.
4. Wey S, Mori M, Pfaller M, Woolson R, Wenzel R. Hospital-acquired candidemia. he attributable mortality and excess length of stay. Arch Intern Med. 1988;148(12):2642-5.
5. Karabinis A, Hill C, Leclercq B, Tancrède C, Baume D, Andremont A. Risk factors for candidemia in cancer patients: a case-control study. J Clin Microbiol. 1988;26(3):429-32.
7. Komshian S, Uwaydah A, Sobel J, Crane L. Fungemia
caused by Candida species and Torulopsis glabrata in
the hospitalized patient: frequency, characteristics, and evaluation of factors inluencing outcome. Rev Infect Dis.1989;11(3):379-90.
8. Ruiz L, Sugizaki M, Montelli A, Matsumoto F, Pires M, da Silva B, et al. Fungemia by yeasts in Brazil: occurrence and phenotypic study of strains isolated at the Public Hospital, Botucatu, São Paulo. J Mycol Med. 2005;15(1):13-21.
9. Beck-Sagué C, Jarvis W. Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980-1990. National Nosocomial Infections Surveillance System. J Infect Dis. 1993;167(5):1247-51.
10. Kurtzman CP, Fell JW. he yeasts: a taxonomic study. 4th ed. Amsterdam: Elsiever; 1998.
11. Weinstein M, Towns M, Quartey S, Mirrett S, Reimer L, Parmigiani G, et al. he clinical signiicance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults. Clin Infect Dis. 1997;24(4):584-602.
12. Edwards JJ, Bodey G, Bowden R, Büchner T, de Pauw B, Filler S, et al. International conference for the development of a consensus on the management and prevention of severe candidal infections. Clin Infect Dis. 1997;25(1):43-59.
13. Ascioglu S, Rex J, de Pauw B, Bennett J, Bille J, Crokaert F, et al. Deining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin Infect Dis. 2002;34(1):7-14.
14. Berenguer J, Buck M, Witebsky F, Stock F, Pizzo P, Walsh T. Lysis-centrifugation blood cultures in the detection of tissue-proven invasive candidiasis. Disseminated versus single-organ infection. Diagn Microbiol Infect Dis. 1993;17(2):103-9.
15. Costa AR, Silva F, Henriques M, Azeredo J, Oliveira
R, Faustino A. Candida clinical species identiication:
molecular and biochemical methods. Ann Microbiol. 2010;60(1):105-12
16. Cárdenes-Perera CD, Torres-Lana A, Alonso-Vargas R, Moragues-Tosantas MD, Pontón-San Emeterio J, Quindós-Andrés G, et al. Evaluation of API ID 32C and VITEK-2 to identify Candida dubliniensis. Diagn Microbiol Infect Dis. 2004;50(3):219-21.
17. Sanglard D, Odds F. Resistance of Candida species to
antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis. 2002;2(2):73-85. 18. Pfaller M, Diekema D, Messer S, Boyken L, Hollis
R. Activities of luconazole and voriconazole against
1,586 recent clinical isolates of Candida species
determined by Broth microdilution, disk difusion,
and Etest® methods: report from the ARTEMIS Global
